SEPTEMBER 15. 1938
AKALYTICAL EDITIOK
It is evident that salt progressively migrates without evaporation. It was found t h a t the rate of chloride migration from the ink strokes was greatly increased by the addition of sodium chloride, and to a lesser extent by hydrochloric acid. The hydrochloric acid image appears, however, to fade rapidly, largely disappearing from the paper. Some of the results are shown in Figure 1, which speaks for itself. The third line from the bottom was written with an ink containing 0.5 per cent of sodium chloride, or about 0.30 per cent of added chloride. The image is much heavier than that in the eighth and ninth lines from the bottom, Tvhere the ink used contained 1 per cent of 12 S hydrochloric acid, or about 0.426 per cent of added chloride. It appears, therefore, that in the latter case the chloride must be dissipated by some process other than diffusion through the paper; evaporation of the hydrochloric acid seems the only possible explanation.
Effect of Added Chloride Experiments were also made to compare the effects of adding chlorides of sodium, calcium, zinc, copper, and tin. It x a s thought that the more deliquescent chlorides, such as calcium and zinc chlorides, might shoiv a more rapid chloride migration than sodium chloride Previous investigations had shown that the rate of chloride migration was very sensitire to moisture content-for example, samples of writing stored a few inches from a steam-heated radiator showed no detectable chloride migration even after six months. But as far as could be observed, the sodium, calcium, zinc, and cupric chlorides, when added to the ink, gave essentially the same rate of chloride migration. A totally unexpected result v a s found with stannous chloride, which seemed, when added to ink, to give practically no chloride migration froin the ink stroke; the extent of the migration was considerably less than with the untreated ink. The explanation may be as follows: Stannous chloride is
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probably rapidly oxidized to stannic compounds on the paper. There are only two added chloride atoms for each tin atom, so that a basic stannic chloride is probably formed. Such a substance may well bind the chloride firmly enough essentially to prevent its diffusion. But on addition of silver nitrate, silver chloride is probably formed, so that a silrer image is developed by the chloride test wherever there has been insoluble basic stannic chloride. An alternative explanation may be that the tin remains as nondiffusible, stannous compounds, which reduce silver nitrate directly and give a spurious “chloride” test. The paper was therefore carefully observed after immersion in the silver nitrate, hut before developing. S o blackening TT as seen at this stage, showing that the image frclm the stannous ink must have been a genuine chloride image; the silver had been firit precipitated as silver chloride and not as silver metal. It therefore appears that stannous chloride, added to the ink, definitely s l o m the rate of migration of the chloride in the paper.
Conclusion Since so many factors are concerned in the chloride test for age of inks, any conclusions regarding age of writing, as determined by this test, should be viewed with extreme suspicion. Inks used by federal offices and in banks should contain a definitely known amount of sodium chloride. This vould not only aid in readily identifying the ink, but would also make it possible to determine something of the time element.
Literature Cited (1) Cornish, R . E.,Finn, John, Jr., and hIcLaughlin, William, IND. ENQ.CHEY.,News Ed., 12, 315 (1934). (2) hlesger, O., Rall, H., and Hess, IT., Arch. H-~imind.,92, 108 (1933). R E C E I Y E D ‘ J U ~1, ~
1938.
Evaluation of the Vitamin A Potencv of Feeds G. S. FRAPS Texas Agricultural Experiment Station, A. & M. College of Texas, College Station, Texas
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HE most important values of commercial feeds are their productive energy, digestible protein, and bulk or volume. Fraps (4, 7 ) has shown that the prices of unmixed feeds are, in general, related to their productive energy and digestible protein; with bulky feeds, such as hays, bulk or volume also has a cost. Some feeds have additional values on account of special constituents. These include alfalfa meal and alfalfa-leaf meal on account of vitamin A potency, milk products on account of vitamin G, and bone meal on account of calcium and phosphorus. The fact that domestic animals under some conditions, do no receive enough vitamin A has recently been recognized. Growing chickens and laying hens are the most frequent sufferers in this respect (26, 27, 28). Milk coivs mag suffer from vitamin A deficiency --hen fed on low-grade roughage (6). Other animals may also receive insufficient vitamin A, when fed on restricted rations, as when the pastures have dried up. Even when fed sufficient quantities to maintain good production, hens may not receive enough vitamin A to produce eggs of high vitamin A potency and milk cows may not receive enough to produce milk or butter fat high in vitamin A potency (5, 27, 28).
Recognition of these deficiencies and desire to correct them have led to demands for information regarding the vitamin A potency of feeds, and methods for evaluating suitable carriers of vitamin A potency. Biological methods for estimating vitamin A potency, such as the Sherman-hlunsell method, the U. S. P. method, and the single-dose method, are expensive, require considerable time, and are not highly accurate. They are suitable for special purposes but not practical for the commercial evaluation of animal feeds.
’C-itaminA Potency Vitamin A potency may be due either t o vitamin A, a colorless substance, or to carotene or cryptoxanthin, yellow substances. Carotene seems to be changed to vitamin A by animals and stored as such, for the most part in the liver. Vitamin A as such is not present in animal feeds unless fish liver oils or their concentrates have been added, and when so added may be almost entirely destroyed in 4 weeks (6). The loss of added vitamin A has been studied by Fraps and Kemmerer through absorption of ultraviolet light at 328 mu as measured in a spectrograph ( 6 ) . This is not at present a practical method of evaluating the \-itamin A
content of feeds. ,111 feeds examined contained substances which absorbed light a t the same lvave length as vitnniin -4, 328 mu, and are called for convenience pseudo-vitamin -4. Corrections for the pseudo-x-itamin A may be made by cornparing a sample to which no additions have been made n i t h the sample to xhich the fish liver oil has been added. On account of possible variations of pseudo-vitamin A in different samples of the same feed, this method is not a t present suitable for evaluating the vitamin A added to feeds in the form of fish liver oils or their concentrates. Vitamin A potency of feeds now appears to be derived entirely from carotene (2, 8, 12, 2.5, 29, SO), with the exception of yellow corn, in which cryptoxanthin, closely related to carotene, is a source of vitamin A potency. Of tlie three carotenes, a, /3, and -/, the P-carotene is the most widely distributed (13, 38,36). According to Kuhn and others, little or no a-carotene occurs in grasses (15,17). The bulk of the carotene mTas found by hIacKinney (16) to be 8-carotene in 59 plant species. The Titamin A potency of feeds may therefore be evaluated by estimating their carotene and cryptoxanthin content.
Estimation of Carotene
A method for estimating carotene was presented by Schertz in 1923 (25). Guilbert (11) and the Bureau of Dairy Industry, U. S. Department of Agriculture ( I S ) , have proposed methods especially adapted to feeds. Modifications of the Guilbert method have been proposed by Peterson, Hughes, and Freeman (19) and b y Fraps and Kemmerer (6, 18). Other methods have been suggested (14, 20, 22, 23, 35). Methods have been proposed for carotene in flour (5). Clausen and McCoord (1) for biologica! samples use a single distribution between liquid phases to separate the carotenoids. As associate referee on carotene for the Association, Official Agricultural Chemists, Munsey is studying the methods for carotene, which are described in detail in his first report ( I S ) . In the Guilbert method, the feed is refluxed with saturated alcoholic potassium hydroxide, cooled, and extracted with ethyl ether. The ether is separated from the alcohol by additions of water, and the aqueous solution is extracted with ether. The ether is distilled off and the carotene and xanthophyll are dissolved in petroleum ether. The xanthophyll is removed by several extractions with ethanol, and the petroleum ether is washed with water, dried with anhydrous ssdium sulfate, concentrated, and made up to volume. In the Peterson-Hughes modification, the ethyl ether is not used but the petroleum ether is added directly to the alcoholic extract. The Dairy Industry method differs from the Guilbert method chiefly in direct extraction of the feed iyith alcohol and petroleum ether without the use of the potassium hydroxide, and is more tedious. The Hughes modification of the Guilbert method seems most promising at the present time, although some prefer the Fraps-Kemmerer modification ( 6 ) . The yellow color of the final carotene solution may be read in a colorimeter by comparison against 0.1 or 0.06 per cent potassium bichromate or a dye solution, or in a photoelectric colorimeter, or the density of absorption of light a t 450, 470, and 480 mu may be read in a spectrophotometer ( I S ) . Reading in a colorimeter against bichromate appears to offer a convenient and rapid method for routine tests, especially where more expensive equipment is not available. Reading the solution in a photoelectric colorimeter with suitable filters may possibly offer a method just as rapid and a little more accurate than a visual colorimeter. The spectrophotometer should be more accurate than the colorimeter, especially when other coloring materials are present besides carotene. The yellow coloring matter extracted and purified by the methods referred to above is not always pure carotene. Shinn et al. (29) showed that carotene preparations may contain other coloring matter besides carotene, especially when the solution was prepared from hay and silage of poor quality. Miller (17) found that some carotene may be removed with
tlie xanthophyll n-aslied out with methanol and some xaiitliopliyll (about 5 per cent) may remain with the carcitelie. Fraps and Kemmerer found that the excrement of both rats and chickens contains a yellow pigment, not carotene, el-eii though the animals were fed on rations practically free froni carotene. These yellow pigments act like carotene in the chemical procedure, but have absorption curves different from that of carotene. The excretion of over 100 per cent of the carotene fed to cows reported by Whitnah et al. (3.5) is probably due to yellow coloring materials not carotene. Nethods of purifying carotene solutions by selective absorption on magnesium oxide, magnesium hydroxide, or other adsorbents may be developed (89, 31, 33). However, according to Gillam et al. (9, I O ) , carotene may be isomerized by chromatographic adsorption on a.lumina with production of neo-a-carotene. Measurements of the absorption of light a t different wave lengths by the carotene solutions are being undertaken by means of high-power spectroscopes. Using a high-power monochromater, with 4 prisms, it is possible to separate absorption bands of light not separable by less powerful instruments. Hogness et al. (17, 37) devised a n apparatus which uses a photoelectric cell, and Miller (17) used this instrument in the study of Carotenoids. Such an instrument may be used in the estimation of carotene in the presence of other coloring materials. One is being installed a t the Bureau of Dairy Industry, Ti. S.Department of Agriculture a t Beltsville, Md., and another has been ordered for the Indiana Experiment Station a t Lafayette, Ind. The high cost of the special equipment required mill necessarily confine this spectroscopic method to a few laboratories, but its use may provide correction factors for results obtained on corresponding samples by other methods and help to make the estimation of carotene more accurate. The relation between the carotene content and the biological potency of a number of animal and human foods is being ascertained a t the Texas dgricultural Experiment Station. Development of methods for determining carotene enable digestion experiments to be conducted, some of the results of which 1%-erereported a t the Dallas meeting of the AMERICAN CHEMICAL SOCIETY.The possible destruction (Jf carotene by fermentation or conversion of xanthophyll or other coloring matters into substances which resemble carotene, by the acids in the gastric juice or other digestive processes, remain to be explored (21). The estimation of the color of hay, as in the commercial grading of hay, is to a certain extent an evaluation in terms of carotene. H a y graded KO. 3 in color is low in carotene; S o . 2 should contain more carotene than N o . 3, and S o . 1 on an arerage should be higher in carotene than S o . 2 or KO.3. Hay may have the good color of grade To. 1 and yet, not be high in carotene, because the chlorophyll is more resistant t o change than the carotene. Grading of hay for carotene by color is not an exact method but, until short methods are developed for use in commercial grading, the color of hay is of import,ance in considering its quality when vitamin A potency is needed. The importance of a high carotene content in alfalfa-leaf meal and some other feeds has been commercially recognized. Feeds are being purchased on minimum specifications for carotene in localities as widely separated as S e w York and California, and analyses are being made to see that the carotene content comes up t o specifications. The demand for high-carotene feeds will no doubt lead to improrernerit' in the methods of curing and preserving such hays, so as to maintain a high carotene content. The vitamin A potency of feeds can therefore be evaluated by the estimation of carotene and cryptoxanthin. Methods for estimating carotene are available, and are being studied and improved. Their accuracy a t the present time is proh-
SEPTEMBER 1.5, 1938
-4NALI-TICAL EDITION
ably a s great or greater than the biological methods of r a t assay.
Literature Cited (1) Clausen, S. IT., and hlcCoord, A. B., J . B i d . Chem., 113, 89
(1936). (2) Escudero, d.,Act. trab. I‘ congr. nac. m k f . , 4, SO0 (1934); Anales asoc. qtiim. orgentina, 24, 11R (1934). (3) Ferrzri, C. G., Cereal Chem., 10, 277-86 (1933). (4) Fraps, G. S.,Texas h g r . Expt. Sta. BulZ. 323 (1924). ( 5 ) Fraps, G. S., Copeland, 0. C., Treichler, Ray. and Kemmerer, A. R.. Ibid., 536 (1937). (6) Fraps, G. 9.. and Kemmerer, A. R., Ibid., 557 (1937). (7) Fraps, G. S., Kemmerer, A. R . , and Fuller, F. D., Texas Bgr. Expt. Sta. Control Circ. G . (1935). (8)Fraps, G. S., Treichler, R., and Kemmerer, A. R.. J . Agr. Research, 53, 713-16 (1936). (9) Gillam, -4.E., and Ridi, M. S. El, Biochem. J . , 30, 1 7 3 5 1 2 (1930). (10) Gillam, A . E., Ridi, bl. S. El, and Kon, 9. K., Ibid., 31, 160510 (1937). (11) Guilbert, H. P., ISD. ESG. CHEW,Anal. Ed., 6, 452 (1934). (12) H a r t m a n , A. hI., Kane, E. , I . , and Shinn, L. A , J . B i d . Chem., 105, 30 (1934). (13) Karrer, P . , and Schlientz, W., Helv. Ciaim. Acto, 17, 7 (1934). 114) Iiropis. A , , Biochem. Z.,287, 226-34 (1937). (15) Kuhn, R., and Lederer, E., 2. physiol. Chem., 200, 246 (1931). (16) MacKinney, G., J . R i d . Chem., 171, 75-84 (1935). (17) Miller, E . S., J . Am. Ciienz. SOC.,57, 347 (1935). (18) blunsey, V. E., J . Assoc. Oficial Agr. Chem., 20, 459-65 (1937). (19) Peterson 11.. J., Hughes, J. S., and Freeman, H. F . , ISD. ESG. CHEM., h a l . E d . , 9, 71-2 (1937).
A Greaseless Stopcock L . S. ECHOLS. JR. Priiic-rton U n i > e r c i t j . Princeton. V.
J.
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(20) Pyke, AI., J . Soc. Chem. I n d . , 55, 139-403 (1936). (21) Quackenbush, F. W., Steenbock, H., and Peterson, TT. H., J . B i d . Chem. (Proc.), 123, xcviii (1938). (22) Ratchei-sky, P., BdZ.S O C . chiin. bzol., 17, 1187-93 (1935). (23) Reti, I,., and Arias da Silva, C.. Anales aso~:.qitim. argentina. 24, 20B (1936). (24) Russell, W.C., Taylor, M.IT., and Chichester, D. F., Proc. SOC. Erptl. Bid. N e d . , 30, 376 (1932): Ezpt. SLa. Record, 68, 863 (1932). (25) Schertz, F. XI., J . A g r . RePearch, 26, 383 (1923). Texas Agr. Expt. Sta. Bull. (26) Sherwood, R. bl.. and Fraps, G . S., 493 (1934). (27) Ibid., 514 (1935). (28) Ihid., 528 (1936). (29) Shinn, L. A., Kane, E. A, Wiseman, H . G., and Cary, C. A , , J . B i d . Chem. (Proc.), 119, lxxxix (1937). (30) Shinn, L. A , Kane, E. rl.. Wiseman, H. G . , and Cary, C. A., Proc. A m . S O C .Animal Production, 27, 1934, 190-2. (31) Strain, H. H.. J . B i d . Chem., 105, 523 (1934:’. (32) Ibid., 111, 85-93 (1935). (33) Strain, H . H., Science. 79, 325 (1934). (34) K h i t n a h , C. H., Peterson, IT. J., iltkeson, F. W., and Cave, H. W., “Carotene Excretion by Dairy Cows,” presented a t Rochester meeting of American Chemical Society, 1937. (35) Wiseman, H . G., and Kane. E. A . , J . B i d . Chem., 114, 108 (1936). (36) Zechmeister, L., “Carotinoide,” Berlin, Julius Springer, 1934. (37) Zscheile, Hogness, and Young, J . P h y s . C‘hem., 38, 1 (1934). RECEIVED April 12, 1935. Presented before the Division of Biologioa Chemistry at the 96th Meeting of the American Chemical Society, Dallas, Texas, April 18 t o 22, 1935.
Several of these stopcocks have been used, still nith the original lubricant, in the Triter’s laboratory for a period of 3 years, during which time they 1i:tr.e required no care whatever. They are positive in action, insensitive t o temperature changes. and completely eliminate the hysteresis effects inherent in the grease-lubiicatetl type.
HILE investigating the decomposition of a number of hydrocarbons and other organic vapors such as the all
